Project Summary Damage to the genetic material of cells can result in tissue dysfunction. The nervous system is particularly vulnerable to genomic instability, and the accumulation of neuronal DNA damage, particularly DNA double strand breaks (DSBs), is an early pathological event observed in Alzheimer?s Disease patients. In the CK-p25 mouse model of severe neurodegeneration, the accumulation of DSBs precedes the appearance of all other pathologies, including neuronal loss and cognitive decline. This suggests DSBs are an initiating lesion of neurotoxicity in these mice. Pathological levels of DSBs are known to induce type I interferon and antiviral response. It is unclear if DSBs initiate innate immune signaling in neurons, and if this plays a role in the hallmark neuroinflammatory cascade associated with neurodegenerative disease. The goal of this proposal is to test if hallmark neuroinflammatory activity is initiated by consequence of DSB-induced innate immunity in neurons. We first established DSBs are sufficient to drive type I interferon and antiviral gene expression in primary neuronal culture, and that this is dependent upon Stat1 activity, a transcription factor that drives the antiviral Jak/Stat signaling pathway. We used fluorescence-activated nuclei sorting to isolate neurons in the CK-p25 cortex with increased immunoreactivity for ?H2AX, a robust marker for DSBs. Transcriptomic analysis revealed ??H2AX-positive? neurons are enriched for innate immune and antiviral pathways. We hypothesized these ?H2AX-positive neurons use innate immune signaling to initiate a neuroinflammatory response in glial cell types. Cell type specific transcriptional analysis of astrocytes, microglia, and oligodendrocytes revealed microglia have the strongest glial inflammatory response at this time-point. This suggests neurons harboring many DSBs may be signaling innate immune distress signals to microglia during the early stages of neurodegenerative disease. The first aim of this proposal will test the hypothesis that DSB-mediated type I interferon and antiviral activity from neurons signals microglial inflammation, and that this is dependent upon neuronal Jak/Stat signaling. This aim will be achieved through DNA DSB induction in primary neuronal culture, followed by co- culture with microglia. The second aim of this proposal will determine if innate immune signaling stemming from ?H2AX-positive neurons activate inflammatory signaling in microglia in vivo. This aim will be achieved through viral knock down of Stat1 in neurons in the CK-p25 forebrain. I will utilize nuclei sorting to assess cell type specific expression of inflammatory genes in both ?H2AX-positive neurons and microglia. To validate changes in the microglial inflammatory response, I will use immunohistochemistry to assess microglial morphology. This proposal will evaluate the impact of DSB-induced innate immune signaling in the initiation of neuroinflammation associated with neurodegeneration, potentially providing new avenues for therapeutic intervention.